Nobuhiko Okada

Increased Red Blood Cell Distribution Width Associates with Cancer Stage and Prognosis in Patients with Lung Cancer

Background Red cell distribution width (RDW), one of many routinely examined parameters, shows the heterogeneity in erythrocyte size. We investigated the association of RDW levels with clinical parameters and prognosis of lung cancer patients. Methods Clinical and laboratory data from 332 patients with lung cancer in a single institution were retrospectively studied by univariate analysis. Kaplan-Meier survival analysis and Cox proportional hazard models were used to examine the effect of RDW on survival. Results The RDW levels were divided into two groups: high RDW (>=15%), n=73 vs. low RDW, n=259 (<15%). Univariate analysis showed that there were significant associations of high RDW values with cancer stage, performance status, presence of other disease, white blood cell count, hemoglobin, mean corpuscular volume, platelet count, albumin level, C-reactive protein level, and cytokeratin 19 fragment level. Kruskal-Wallis tests revealed an association of RDW values with cancer stage in patients irrespective of comorbidity (patient with/without comorbidity: p<0.0001, patient without comorbidity: p<0.0001). Stages I-IV lung cancer patients with higher RDW values had poorer prognoses than those with lower RDW values (Wilcoxon test: p=0.002). In particular, the survival rates of stage I and II patients (n=141) were lower in the high RDW group (n=19) than in the low RDW group (n=122) (Wilcoxon test: p<0.001). Moreover, multivariate analysis showed higher RDW is a significant prognostic factor (p=0.040). Conclusion RDW is associated with several factors that reflect inflammation and malnutrition in lung cancer patients. Moreover, high levels of RDW are associated with poor survival. RDW might be used as a new and convenient marker to determine a patient’s general condition and to predict the mortality risk of lung cancer patients.

Virulence‐associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis

Shigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the IpaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 Notl restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence‐associated chromosomal loci.

Complete DNA Sequence and Comparative Analysis of the 50-Kilobase Virulence Plasmid of Salmonella enterica Serovar Choleraesuis

ABSTRACT The complete nucleotide sequence of pKDSC50, a large virulence plasmid from Salmonella enterica serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the spv operon, which is composed of genes essential for systemic infection by nontyphoidalSalmonella. Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the spvRABCD genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the pef(plasmid-encoded fimbria) operon and rck (resistance to complement killing) gene, which has been identified on the virulence plasmid of S. enterica serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic Escherichia coliO157:H7 and the enteropathogenic E. coli (EPEC) adherence factor plasmid harbored by EPEC strain B171 (O111:NM), as well as the virulence plasmids of Salmonella serovars Typhimurium and Enteritidis. Comparative analysis of the nucleotide sequences of the 50-kb virulence plasmid of serovar Choleraesuis and the 94-kb virulence plasmid of serovar Typhimurium revealed that 47 out of 48 ORFs of the virulence plasmid of serovar Choleraesuis are highly homologous to the corresponding ORFs of the virulence plasmid of serovar Typhimurium, suggesting a common ancestry.

Streptococcus pyogenes protein F promotes invasion of HeLa cells

Although the Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) has been considered an extracellular pathogen which adheres to human mucosal epithelium, the streptococcus possesses invasive capacity for cultured human epithelial cells. This study provides genetic and functional evidence supporting the conclusion that protein F is capable of mediating entry of S. pyogenes into HeLa cells. Using Tn916 insertion mutagenesis or an isogenic S. pyogenes strain with a defined mutation in the gene encoding protein F (prtF), it was observed that the invasive capacity was affected by the levels of surface-exposed protein F, but not by those of M protein. In addition, heterologous expression of protein F on Enterococcus faecalis conferred upon the bacteria an efficient invasive phenotype. Several assays demonstrated that both the fibronectin-binding domains of protein F, UR and RD2, were involved in host-cell invasion. In addition, coinfection experiments of HeLa cells with S. pyogenes and an Escherichia coli K-12 strain expressing an afimbrial adhesin AFA-I showed that the uptake of S. pyogenes did not permit internalization of the E. coli cells.

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection

Salmonella phosphothreonine lyase SpvC inactivates the dual‐phosphorylated host mitogen‐activated protein kinases (MAPK) through β‐elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)‐1 and SPI‐2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI‐2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI‐1 and SPI‐2 T3SSs. Dephosphorylation of the extracellular signal‐regulated protein kinases (ERK) was detected in an SPI‐1 T3SS‐dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild‐type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro‐inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti‐inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.

PROTECTION AGAINST SHIGA TOXIN 1 CHALLENGE BY IMMUNIZATION OF MICE WITH PURIFIED MUTANT SHIGA TOXIN 1

ABSTRACT Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene.

ABSTRACT Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.

Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Extracellular secretion of the virulence plasmid-encoded ADP-ribosyltransferase SpvB in Salmonella.

Nontyphoid Salmonella enterica requires the plasmid-encoded spv genes to establish successful systemic infection in experimental animals. The SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain. SpvB ADP-ribosilates actin and depolymerizes actin filaments when expressed in cultured epithelial cells. However, spontaneous secretion or release of SpvB has not been observed under in vitro growth conditions. In the present study we investigated the secretion of SpvB from Salmonella using in vitro and in vivo assay systems. We showed that SpvB is secreted into supernatant from Salmonella strains that contain the cloned spvB gene on a plasmid when they grew in intracellular salts medium (ISM), a minimal medium mimicing the intracellular iron concentrations of eukaryotic cells. A series of mutant SpvB proteins revealed that an N-terminal region of SpvB located at amino acids 1-229 was sufficient to promote secretion into extracellular milieu. Confocal immunofluorescence microscopy also demonstrated efficient localization of the N-terminal domain of SpvB(1-360) tagged with biotinylated peptide within infected host cell cytosol but not truncated SpvB(1-179) fusion protein. In addition, mutations that inactivate genes within Salmonella pathogenicity island 1 or Salmonella pathogenicity island 2 that encode type III secretion systems (TTSS) could secrete the SpvB protein into the culture medium. These results indicate that SpvB protein is transported from the bacteria and into the host cytoplasm independent of TTSS.

Oral Administration of Heat-Killed Lactobacillus plantarum Strain b240 Protected Mice against Salmonella enterica Serovar Typhimurium

The purpose of this study was to investigate the effects of heat-killed Lactobacillus plantarum strain b240 (b240) on systemic infection by Salmonella enterica serovar Typhimurium (S. Typhimurium) and to determine the mechanism by which b240 protects against infection. Mice were administered either b240 or saline orally for 3 weeks, and then inoculated with S. Typhimurium. The mice treated with b240 were significantly protected against S. Typhimurium as compared to those fed saline. Moreover, translocation of S. Typhimurium into each organ tested in the mice that received b240 tended to be less than in the control mice. An important mechanism of protection against infection was demonstrated by the ability of b240 to inhibit both binding by and invasion of S. Typhimurium into cells. These results indicate that nonviable lactic acid bacteria also play important roles in preventing infection by enteric pathogens.